The present invention relates to the identification of auxiliary genes that encode proteins involved in antibiotic resistance in bacteria, and to compounds that can antagonize the activity of such proteins, thereby resensitizing resistant bacteria to antibiotics.
Methicillin resistant strains of Staphylococcus aureus (MRSA) have become first ranking nosocomial pathogens worldwide. These bacteria are responsible for over 40% of all hospital-born staphylococcal infections in large teaching hospitals in the US. Most recently they have become prevalent in smaller hospitals (20% incidence in hospitals with 200 to 500 beds), as well as in nursing homes (Wenzel et al., 1992, Am. J. Med. 91(Supp 3B):221-7). An unusual and most unfortunate property of MRSA strains is their ability to pick up additional resistance factors which suppress the susceptibility of these strains to other, chemotherapeutically useful antibiotics. Such multiresistant strains of bacteria are now prevalent all over the world and the most xe2x80x9cadvancedxe2x80x9d forms of these pathogens carry resistance mechanisms to all but one (vancomycin) of the usable antibacterial agents (Blumberg et al., 1991, J. Inf. Disease (63:1279-85).
A most ominous and recent development is the appearance of a vancomycin resistance mechanism in another nosocomial pathogenxe2x80x94Enterococcus faeciumxe2x80x94which is known for its ability to transfer from one cell to another plasmid-born resistance factors, such as vancomycin resistance. The arrival of vancomycin resistance to MRSA is only a matter of time. Once this happens, an invasive bacterial pathogen without any antibacterial agent to control it will result. This event would constitute nothing short of a potential public health disaster of immense proportion (Leclercg et at., 1988, New Eng. J. Med. 319:157-61).
The preceding explains the intense interest in the public health and pharmacological community in any new method that promises a usable intervention against MRSA. A more complete explanation of the basis for antibiotic resistance follows.
The central genetic element of methicillin resistance is the so called mecA gene. This gene is found on a piece of DNA of unknown, non-staphylococcal origin that the ancestral MRSA cell(s) must have acquired from a foreign source. The mecA gene encodes for a penicillin binding protein (PBP) called PBP2A (Murakami and Tomasz, 1989, J. Bacteriol. 171:874-79), which has very low affinity for the entire family of beta lactam antibiotics. In the current view, PBP2A is a kind of xe2x80x9csurrogatexe2x80x9d cell wall synthesizing enzyme that can take over the vital task of cell wall synthesis in staphylococci when the normal complement of PBPs (the normal catalysts of walt synthesis) can no longer function because thy have become fully inactivated by beta lactam antibiotic in the environment. The critical nature of the mecA gene and its gene product PBP2A for the antibiotic resistant phenotype was best demonstrated by transposon inactivation experiments in which the transposon Tn551 was maneuvered into the mecA gene. The result was a dramatic drop in resistance level from the minimum inhibitory concentration (MIC) value of 1600 xcexcg/ml in the parental bacterium to the low value of about 4 xcexcg/ml in the transposon mutant (Matthews and Tomasz, 1990, Antimicrobial Agents and Chemotherapy 34:1777-9).
This observation is consistent with the foregoing theory. The mutant bacteria with their interrupted mecA gene could no longer synthesize PBP2A; thus the surrogate enzyme needed for the survival in the antibiotic-rich environment was no longer available to catalyze wall synthesis. Consequently, the methicillin susceptibility of the Tn255 mutant dropped to a level approaching the susceptibility of staphylococci without the mecA gene. Methicillin MIC for such bacteria is usually in the vicinity of 1-2 xcexcg/ml.
Additional genetic work resulted in several surprising observations. First it was found that the level of antibiotic resistance could also be dramatically lowered in transposon mutants in which the Tn551 did not interrupt the mecA gene or interfere with the expression of this gene (i.e., the production of PBP2A). Clearly, these mutants were low in resistance for some reason other than an interruption of the functioning of the mecA gene. In fact, it turned out that the great majority of Tn551 insertional mutants with reduced methicillin resistance all continued to produce normal amounts of PBP2A in spite of the fact that their resistance level could be reduced by very large factors, such as dropping from the methicillin MIC of 1600 xcexcg/ml to a low of 3 xcexcg/ml.
The first such mutant was isolated in 1983 by Swiss scientists at a time when the nature of methicillin resistance was hardly understood at all (Berger-Bxc3xa4chi, 1983, J. Bacteriol. 154:479-87). Subsequent work in several laboratories have increased the number of these genetic determinants, the common feature of which was that they had an intact mecA gene and yet they had reduced resistance levels to the beta lactam family of antibiotics. The provisional name xe2x80x9cauxiliary genesxe2x80x9d was proposed for this class of unusual genetic elements to imply that they appeared to perform some essential xe2x80x9chelperxe2x80x9d function(s) in the expression of high level beta lactam resistance (Tomasz, 1990, In Molecular Biology of the Staphylococci, Novick and Skurray, Eds., VHC Publishers: New York, pp. 565-583).
A second surprising observation concerned the number of auxiliary genes that have been identified. By 1993, the number of genetically distinct auxiliary mutants described in the literature had risen to four; presently, six have been identified [Berger-Bxc3xa4chai, Trends in Microbiology, 2:389-392 (1994); DeLencastre et al., J. Antimicrob. Chemother. 33:7-24 (1994); Henze et al., J. Bacteriol. 175: 1612-1620 (1993); Maidhof et al., J. Bacteriol. 173:3507-3513 (1991)].
A third set of observations provided clues as to the biochemical nature of auxiliary functions. It was shown by a newly developed high resolution chromatography technique that many of the auxiliary mutants produced abnormal peptidoglycan in their cell walls. Studies combining High Performance Liquid Chromatography (HPLC) and mass spectrometry allowed the identification of the chemical changes that occurred in the mutants (De Jonge et al., 1991, J. Bacteriol. 173:1105-10; De Jonge et al., 1992, J. Biol. Chem. 267:11248-54; De Jonge et al., 1992, J. Biol. Chem 267:11255-9; and De Jonge et al., 1993, J. Bacteriol. 175:2779-82). The cell wall peptidoglycan of auxiliary mutants was composed of muropeptides (cell wall building blocks) either with incomplete cross-linking peptides or containing a free glutamic acid residue instead of the usual isoglutamine. Still other mutants showed different cell wall muropeptide fingerprints in which the exact nature of changes remains to be elucidated. These findings suggest that the auxiliary genes are genes involved with the biosynthesis of cell wall precursor muropeptides.
While all the numerous auxiliary mutants share the common feature of carrying an intact mecA, each one of the auxiliary genes are unique by the criteria of (i) physical location on the chromosome as determined by restriction mapping; (ii) in the several cases in which DNA sequences of the genes were determined (as in the cases of the auxiliary genes known as femA, femB and femC) (Berger-Bxc3xa4chi et al., 1992, Antimicrobial Agents and Chemotherapy 36:1367-73; Gustafson et al., 1993, In Abstracts of the 93rd General Meeting of the American Society for Microbiology, Abstract A-97, p. 18; and De Lencastre et al., 1993, xe2x80x9cMolecular Aspects of Methicillin resistance in Staphylococcus aureusxe2x80x9d, J. Antimicrob. Chemother. 33:), the genes were shown to have unique DNA sequences; and (iii) in the cases in which the mutants had altered cell wall composition, the HPLC patterns provided additional gene-specific fingerprints characteristic of the particular mutant.
Recently, a new tranposon library constructed in the background of the highly and homogeneously methicillin resistant Staphylococcus aureus (MRSA) strain COL yielded 70 independent insertional mutants with reduced levels of antibiotic resistance, out of which only two were inserts in mecA while the rest were scattered over seven of the sixteen SmaI fragments of the COL chromosome. Preliminary studies suggest that this library includes at least 10 to 12 new genetic determinants, each of which is needed for optimal expression of methicillin resistance [International Patent Publication No. WO 95/16039, published Jun. 15, 1995 by DeLencastre and Tomasz; DeLencastre and Tomasz, Antimicrob. Agents. Chemother. 38:2590-2598 (1994)].
The citation of any reference herein is not an admission that such reference is available as prior art to the instant invention.
The present invention is broadly directed to the identification of a new auxiliary gene encoding a protein associated with antibiotic resistance in bacteria, in particular Gram positive bacteria, to characterizing the phenotype of bacteria having a mutation in this auxiliary gene, and to identifying compounds that can mimic the phenotype of bacteria in which the activity of the auxiliary gene is disrupted.
In a preferred aspect, the invention is directed to a mutant antibiotic-resistant Staphylococcus aureus strain characterized by increased sensitivity to an antibiotic to which a parent of the mutant strain is resistant, and location of the mutation in a SmaI-I fragment of the chromosome of S. aureus. Generally, the antibiotic is a beta lactam antibiotic, in particular, methicillin. In a preferred aspect, the mutation is caused by insertion of transposon Tn551.
In a specific embodiment, characterized by RUSA315, the mutation results in a blockade of cell wall synthesis at or close to the branch point in hexose metabolism involved in the synthesis of cell wall components.
The invention is further directed to a DNA molecule comprising a nucleic acid sequence which encodes a protein associated with antibiotic resistance in a S. aureus bacterium, which nucleic acid sequence is preferably located in the SmaI-I fragment of the chromosome of the S. aureus bacterium. In particular, the DNA molecule comprises in gene, a mutation which results in a blockade of cell wall synthesis at or close to the branch point in hexose metabolism involved in the synthesis of cell wall components. The compositional change of peptidoglycan in a mutant of the invention is the complete disappearance of the unsubstituted disaccharide pentapeptide monomer. In a specific embodiment, the mutant corresponds to RUSA315. In a still further embodiment, the gene has a nucleotide sequence as depicted in FIG. 6 (SEQ ID NO: 1).
The invention is also directed to a recombinant vector comprising the DNA molecule described above, operatively associated with an expression control sequence, and to a bacterial cell comprising the recombinant vector.
In another aspect, the invention is directed to a method for identifying a compound useful for sensitizing bacteria to an antibiotic to which the bacterium is resistant, comprising identifying a compound that antagonizes the activity of a protein associated with antibiotic resistance in a S. aureus bacterium, which protein is preferably encoded by a nucleic acid sequence located in the SmaI-I fragment of the chromosome of the S. aureus bacterium. Preferably, the protein is involved in synthesis of cell wall components derived from hexose. In a preferred aspect of the invention, the composition and structure of the bacterial cell wall can be analyzed by high performance liquid chromatography and mass spectrometry to determine the association of the protein with muropeptide precursor synthesis. In a specific embodiment, the invention relates to identification of a compound the administration of which results in lack of unsubstituted disaccharide pentapeptide monomer in the bacterial cell wall. This embodiment of the invention relates to identification of a compound the administration of which results in lack of hexose-derived cell wall precursors.
In another specific embodiment, the invention contemplates reducing beta lactam antibiotic resistance in bacteria by administration of a competitive inhibitor antagonist of an enzyme or enzymes involved in synthesis of cell wall compounds derived from hexose.
In yet another aspect, the invention relates to a method for treating a subject suspected of having a bacterial infection comprising administering to the subject an amount of a compound useful for sensitizing the bacteria to an antibiotic to which the bacterium is resistant in conjunction with an amount of the antibiotic sufficient to neutralize the bacteria. Preferably, the compound inhibits or antagonizes the activity of a protein associated with muropeptide precursor synthesis, in particular the synthesis of cell wall compounds derived from hexose.
Accordingly, the invention also relates to a pharmaceutical composition for use in treating a subject suspected of halving a bacterial infection comprising a compound in an amount effective to sensitize bacteria to an antibiotic to which the bacteria are resistant and a pharmaceutically effective carrier. In a further embodiment, the, pharmaceutical composition also comprises an antibiotic in an amount sufficient to neutralize the bacteria.
Although not intending to bound by any particular mechanistic theory or hypothesis, the inventors believe that in the presence of a beta lactam antibiotic the drug molecules and molecules of the cell wall building blocks (muropeptides) compete for the active site of PBP2A, i.e., the surrogate enzyme that, under these conditions, is solely responsible for cell wall biosynthesis. Intact, functioning auxiliary genes allow the production of all the normal cell wall precursor muropeptides, which are highly effective in the competition for the enzyme active site. Thus, in such a staphylococcal cell, relatively higher concentration of the antibiotic is needed for the inactivation of PBP2A, driving the antibiotic MIC value up.
In contrast, inactivated auxiliary genes may prevent the formation of structurally normal cell wall precursors in appropriate intracellular concentrations. Such structurally abnormalxe2x80x94or concentration-wise inadequatexe2x80x94cell wall precursors do not have high enough affinity for the active site of PBP2A., Thus, the relative effectiveness of the drug molecules increases, driving the MIC value down.
However, the identification of strains mutated in auxiliary genes which do not appear to be directly associated with muropeptides precursor synthesis suggests that there are interacting cellular pathways involved in antibiotic resistance.
It is a particular advantage of the invention that the compounds of the invention make possible the use of the known battery of antibiotics, rather than requiring development of new antibiotics, for the treatment of bacterial infections.
The primary object of the invention is to identify compounds that reverse antibiotic resistance in bacteria. These compounds can be used in conjunction with the antibiotics to treat bacterial infections not otherwise amenable to chemotherapy.
Thus, it is an object of the present invention to identify auxiliary genes encoding proteins directly or indirectly associated with antibiotic resistance in bacteria.
It is also an object of the invention to identify such auxiliary genes that encode proteins involved with cell wall precursor synthesis.
Yet another object of the invention is to isolate, sequence and characterize such genes, in order to evaluate the functional activity of the protein encoded by the gene.
It is yet a further object to prepare such proteins in purified form for structural and functional analysis.
Most importantly, it is an object of the invention to screen for and select compounds that reverse antibiotic resistance of bacteria.
These and further objects of the invention will become more clear after consideration of the following FIGURES and DETAILED DESCRIPTION.